Insulin initiates its effects on metabolism by binding to specific receptors on the surface of cells. For insulin in blood to react with these receptors in vivo, the hormone must cross the capillary endothelium and extracellular space to reach the cells. Recent studies by others in cultured endothelial cells indicate that insulin may be transported across capillary endothelial cells by a receptor-mediated process. We report here a study of transport of insulin across capillary endothelium in perfused rat adipose tissue. Since we were unable to measure directly the concentration of insulin in the extravascular fluid, we measured transport of insulin across endothelium by the effect of intraarterially infused insulin on oxidation of (U-14C) glucose to CO2. Glucose oxidation was constant in adipose tissue perfused with 0 to 50 microunits of insulin per ml. The rate of oxidation was doubled in 90 min at 100 microunits/ml, and maximal in 40 min at 200 microunits/ml and maximal in 20-30 min at 500 microunits/ml. The slow decline in oxidation rate when insulin infusion was stopped suggests that insulin was sequestered in the tissue. The half-maximal response to insulin occurred at a much higher insulin concentration in perfused tissue than in incubated adipocytes and incubated adipose tissue, and the time required for maximal response was longer in perfused adipose tissue. The data indicate that transfer of insulin from blood to parenchymal cells in perfused adipose tissue was restricted. The minimal amount of insulin needed for a response by adipocytes in perfused tissue was estimated to be less than 1% of that in blood. Our findings are consistent with the concept that insulin is transferred across capillary endothelium by a receptor-mediated process.